| Foodborne diseases is harmful to human public health, Staphylococcus aureus and Escherichia coli0157:H7are the main pathogenic bacteria.Staphylococcus aureus causes many serious infections, it is an important pathogen which can cause suppurative infection of animal and human, and it is one of the common pathogenic bacteria that can cause human food poisoning. In our country, food poisoning which caused by Staphylococcus aureus is accounted for about25%, in the United States, the number is up to33%, while in Canada the number is more than45%.Escherichia coli is located in the.gut of human and animal. Most of them are not pathogenic, but certain serotypes are pathogenic strains, such as intestinal tract enterohemorrhagic Escherichia coli (EHEC). EHEC is important cause of foodborne illness, and the E.coli0157:H7is the most common cause of human disease serum type. In1982, Escherichia coli O157:H7was first confirmed that it can infect human in the United States, and after then more than80countries worldwide reported that Escherichia coli0157:H7infected people, United States has the highest incidence, the route of infection was mainly through contamination of food and water. In1999the outbreak of E.coliO157:H7infection, caused the death of83people in Jiangsu Xuzhou of China. These two kinds of pathogenic bacteria often appear at the same time in animal foods, testing these two kinds of bacteria can effectively control the propagation and reduce its threat to people.Traditional biochemical separation identification method for detecting the presence of a detection cycle is long, the reagent,low detection limit, need to have identification technology professionals to participate in.The traditional detection methods are not fit in today’s rapid economic development, so to establish a quick, accurate, convenient detection method is particularly important.Liquid-chip technology is developed in recent years, it can be used in the detection and many other areas, high specificity, high sensitivity, high throughput and flexibility etc makes the liquid chip technology gradually entering clinical diagnosis field. This study is the two pathogens of monoclonal antibodies as capture antibody and microsphere carrier coupling, the polyclonal antibody as detecting antibody biotin-antibodies as the second detection antibody, SA-PE as report to establish liquid phase chip detection system.The research results are as follows:1.Optimized the condition of liquid chip methods for detection of Escherichia coli O157:H7, the condition:the working concentration of polyclonal antibody was1:100, biotin labeled IgG was1:500, SA-PE was1μg/mL, time was30min。The sensitivity of detection was104CFU/mL, in the scope of selected similar foodborne pathogens the detection specificity was100%; coefficient of variation was3.86%.2.Optimized the condition of liquid chip methods for detection of Staphylococcus aureus, the condition:the working concentration of polyclonal antibody was1:100, biotin labeled IgG was1:1000, SA-PEwas2μg/mL, time was40min。The sensitivity of detection was103CFU/mL, in the scope of selected similar foodborne pathogens the detection specificity was100%; coefficient of variation was8.31%.3Based on the results of the study were established liquid-chip method for simultaneous detection of two pathogens methods,to detect the sensitivity, specificity, reproducibility, and artificial infect samples to determine the limit of this method, and the application of the method of200actual samples testing, test results were as follows:sensitivity of these two kinds of pathogenic bacteria were same as the single method results; in the scope of selected similar foodborne pathogens specificity was100%; good stability by artificial infection test; the limit of E. coli O157:H7contamination in food samples was10CFU/mL,the Staphylococcus was50CFU/mL.The result of developed method correspond to national standard method in the actual sample testings but there was certain false positive.Experiments prove that the method developed in this study, fast, simple, sensitive, good stability, in the inspection and quarantine has broad development prospect... |
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